The project is directed toward identification of proteins of the anthrax pathogen, Bacillus anthracis, that can be used as targets for novel antibiotics and antibiotic potentiators. The approach to be taken is to use a powerful genetic strategy, signature tag mutagenesis (STM), to identify bacterial genes whose disruption prevents the pathogen from undergoing certain specific stages of the infection process. These include genes critical for germination of spores within macrophages, survival and proliferation of bacteria within macrophages, their escape from macrophages, survival and proliferation of bacteria in the human blood, and secretion of toxin molecules. Inhibitors of the protein products of these genes would be able to cause the same effects as genetic disruption and can potentially be used as novel anti-anthrax drugs. These inhibitors will be developed in other projects of this collaborative program. Additionally, strains of B. anthracis resistant to ciprofloxacin, the first-line antibiotic recommended for anthrax treatment and prevention, will be selected, thus mimicking the resistant strains that can be developed by terrorist entities. The STM strategy will then be used to identify genes critical for maintaining the resistance phenotype of these strains. Inhibitors of the protein products of these genes will be useful as potentiators of ciprofloxacin if artificially created resistant strains are ever used as biological weapon. The modified variant of STM that will be employed in this study includes several new features: transposome method of delivery of the tagged marker gene, unusually long (80 bp) tag sequences, and the use of DNA microarray hybridization as a method to reveal negatively selected library clones. This project will reveal new information about the mechanisms of B. anthracis pathogenesis and drug resistance and validate the use of STM for the discovery of new bacterial drug targets. Most importantly, it will provide other projects of this program with information about multiple potential drug targets, thus contributing to the development of new anti-anthrax drugs, as well as drugs that can be used against other bacterial pathogens.